MPT64 antigen-induced immune responses as a novel diagnostic tool for tuberculosis

and their immunogenicity was assessed by enzyme-linked immunosorbent assay (ELISA) in immunized mice. MPT64 emerged as the most potent secretory protein, inducing the highest interferon-gamma (IFN-γ) production in mouse spleen cells. Both MPT64 alone and in combination with CLE (a fusion protein of early secretory antigenic target-6 [ESAT-6] and culture filtrate protein-10 [CFP-10]) significantly enhanced IFN-γ production in peripheral blood mononuclear cells (PBMCs) from TB patients, demonstrating high diagnostic sensitivity and specificity. TB patients exhibited markedly elevated MPT64-specific antibodies, with the immunoglobulin G4 (IgG4) subclass showing optimal detection performance. Skin tests successfully differentiated mice immunized with H37Rv from those immunized with BCG and phosphate-buffered saline (PBS), while immunized rabbits produced high levels of MPT64 antibodies. The peptide TDPLPVVFPIVQGE from MPT64 elicited the strongest IFN-γ production and skin reaction, while TQAVVLKVYQN showed the strongest antibody binding. These results indicate that MPT64 is a specific M. tuberculosis antigen with significant potential for TB diagnosis through multiple approaches, including IFN-γ release assays, skin tests, and serological detection, particularly when combined with CLE for enhanced diagnostic performance. IMPORTANCE: MPT64 is a protein from the tuberculosis (TB) bacterium that triggered the strongest immune response among seven new antigens tested. It shows great promise for making TB diagnosis more accurate. MPT64 worked well in different test formats, including blood tests that measure a key immune molecule (interferon-gamma), tests that detect TB antibodies, and skin tests like the TB skin test. It also performed strongly when combined with another TB antigen fusion called CLE (a merger of two other TB proteins, early secretory antigenic target-6 [ESAT-6] and culture filtrate protein-10 [CFP-10]). These results suggest that adding MPT64 to current TB tests could help doctors detect TB infection more easily and reliably.